Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Micropropagation of the bromeliad Guzmania ‘Hilda’ via organogenesis and the effect of α-naphthaleneacetic acid on plantlet elongation

This study established a highly effective micropropagation system to obtain good plantlet proliferation from floral organs via callus induction and bud differentiation in Guzmania ‘Hilda’ bromeliad. The best frequencies of organogenic callus formation (20% in petal and 35% in ovary explants) were obtained on media containing a combination of 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.5 mg l⁻¹ 2,4-D with 0.5 mg l⁻¹ NAA, respectively. Organogenic calli were cultured on medium with 1.0 mg l⁻¹ NAA and 0.5 mg l⁻¹ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) induce the differentiation and regeneration of adventitious buds into plantlets. When the plantlets were cultured in a medium with optimum NAA concentration (0.5–1.0 mg l⁻¹) significant improvement in regeneration and elongation was achieved within one month. This overcame the difficulty of delayed elongation in Guzmania plantlets. More than 99% of the regenerated and acclimatized plantlets developed to the flowering stage.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Restoration of rooting competence in a mature plant of Garcinia indica through serial shoot tip grafting in vitro

Apical and axillary buds from a high yielding, early fruiting elite tree (more than 20 years old) were cultured in woody plant medium (WPM) supplemented with 0.9 μM N⁶-benzyl adenine (BA). Multiple shoots were obtained on WPM basal medium containing 8.9 μM BA and 0.5 μM thidiazuron (TDZ). Elongation of axillary shoots was obtained in half-strength WPM medium supplemented with 0.4 μM BA. For root initiation, the elongated shoots were transferred to half strength WPM basal medium containing 2.5–245 μM indole-3-butyric acid (IBA) or 2.7–268.5 μM α-naphthaleneacetic acid (NAA) or the shoots were subjected to 2.5–53.9 mM IBA, 2.7–59.1 mM NAA dip for (30 s–30 min) and then transferred to half strength WPM basal medium. However, rooting was never achieved even after 2 months of culture.     

Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor)

Lanzhou lily (Lilium davidii var. unicolor) is one of the best lilies which are edible in China but the efficient shoot regeneration system has not been developed. The purpose of the present study is to establish an efficient and reproducible protocol for induction of shoots in vitro from L. davidii var. unicolor leaves. Shoot regeneration from in vitro cultured leaves of L. davidii var. unicolor was tested on the 26 media based on NN [Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science 163, 85–87] basal medium, containing different concentrations of thidiazuron (TDZ) in combination with different concentrations of α-naphthaleneacetic acid (NAA). Shoot organogenesis occurred directly from the leaves without forming callus. Shoot regeneration mainly occurred from the cuts across the midvein and the base of the leaf explants. The highest frequency of regeneration (93.3%) and the largest number of shoots per leaf (3.83) were obtained on NN basal medium supplemented with 0.5 mg l⁻¹ TDZ and 1.0 mg l⁻¹ NAA. All the regenerated shoots formed complete plantlets on half-strength MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] basal medium containing 0.1–0.5 mg l⁻¹ indole-3-butyric acid (IBA) with in 30 days, and 92% of the regenerated plantlets survived in the soil. This study will be useful for Agrobacterium-mediated transformation and exploitation of somaclonal variation of Lanzhou lily.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Effect of genotype,explant size,position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l⁻¹ thidiazuron (TDZ), 0.8 mg l⁻¹ adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 2.0 mg l⁻¹ kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l⁻¹ IBA. All plantlets acclimatized well in the greenhouse.     

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

Comparison of shoot induction ability of different explants in herbaceous peony (Paeonia lactiflora Pall.)

Shoot induction ability of explants of herbaceous peony was investigated in semisolid MS medium containing BA, TDZ and GA₃. Callus was readily induced from stem without node and petiole explants within 2 days of culture but failed to generate shoots. Adventitious shoots were successfully produced from meristematic regions only: bud eyes on nodal stem sections, and junctions of petioles and petiolules. No shoots were induced from internode sections, petiole without junctions, or leaf sections. Nodal sections were the most efficient explants. There were up to 20 shoots in one explant generated within 20 days of culture. TDZ was more effective than BA to induce shoots. The 100% shoot induction rate was obtained in medium containing 0.1–3 mg L⁻¹ of TDZ. However, higher concentrations of TDZ inhibited shoot elongation and only large leaf clusters were produced. Combinations of BA and TDZ failed to increase shoot induction rates but caused shoots shorter. The 2–60-min pretreatment of explants with 20 mg L⁻¹ TDZ solution was very effective to induce adventitious shoots directly, but both shoot number and shoot length tended to decrease as treatment time increased. GA₃ was beneficial for shoot and stem elongation.     

In vitro shoot proliferation and enhancement of rooting for the large-scale propagation of yellow bamboo (Bambusa vulgaris ‘Striata’)

Continuous and rapidly proliferating axillary shoots were raised from axillary buds in secondary branches of adult field culms and nursery grown 1-year-old tissue culture-raised plants of Bambusa vulgaris ‘Striata’. Shoots continuously proliferated in a MS medium containing 4 mg L⁻¹ 6-benzyladenine (BA). The effects of indole butyric acid (IBA) levels, a pretreatment with thidiazuron (TDZ) (1-phenyl-1-([1,2,3-thidiazol-5-yl])urea) and illumination on rooting, were investigated after 6 months of shoot proliferation. A rooting medium with IBA at 3 mg L⁻¹ was optimum for root induction. Shoots of adult field culms that were proliferated in the presence of BA when induced to root in this medium resulted in 40% rooting in 27 days. In vitro shoots raised from 1-year-old tissue cultured plants showed 92% rooting under the same conditions. Rooting was enhanced when the relatively difficult-to-root in vitro shoots from adult field culms were pretreated with 0.5 mg L⁻¹ TDZ for two to three subcultures before placing in the root induction medium. Continuously illuminated shoots pretreated with TDZ for three subcultures showed 100% rooting compared to 83% rooting of shoots that were exposed to a 12 h photoperiod. These findings have been applied in the large-scale propagation of this species.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

In vitro shoot multiplication and plant regeneration in four Capsicum species using thidiazuron

A procedure of in vitro plant propagation using shoot meristem explants (∼0.5 cm) has been developed for Capsicum annuum cv CA960, C. baccatum, C. frutescens and C. praetermissum on Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497] medium containing various cytokinins. Among various concentrations of cytokinins tested; adenine (Ad), N⁶-benzyladenine (BA), kinetin (Kn), zeatin and thidiazuron (TDZ) individually. TDZ regenerated maximum number (4.2–22.4) of shoots in all the Capsicum species tested. Multiple shoot elongation occurred upon transfer to BA (0.22 μM l⁻¹) + IAA (0.48 μM l⁻¹). Rooting of regenerated shoots was achieved on medium supplemented with 5.71 μM l⁻¹ indole-acetic acid (IAA). Rooting was observed in 72–94% of shoots obtained from TDZ-containing regeneration medium followed by elongation treatment in contrast to 8–22% of shoots without elongation treatment. Plantlets obtained from TDZ-containing media were normal diploid (2n = 24) and could readily be established in the soil under green house conditions with a survival frequency of 68–84%. Regenerated plants were developed into morphologically normal, fertile plants and able to set viable seeds.     

In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.     

Effect of position and orientation of leaflet explants with respect to plant growth regulators on micropropagation of Zamioculcas zamiifolia Engl. (ZZ)

Micropropagation studies on Zamioculcas zamiifolia Engl. (ZZ) as to the position and orientation of leaflet explants and plant growth regulators were carried out. Explants consisted of leaflet lamina from the basal or apical part of the leaflet with or without petiolule or midrib that were placed vertically into the medium except for the explants with midrib from the basal part of the leaflet that were placed horizontally as well. The explants were cultured on solid Murashige and Skoog medium (MS) with 30 g l⁻¹ sucrose, supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2 or 4 mg l⁻¹ and 6-benzyladenine (BA) at 0 or 4.44 μM in all (four) possible combinations, or with 1-naphteleneacetic acid (NAA) at 0 or 5.38 μM and BA at 0 or 4.44 μM in all (four) possible combinations (establishment medium). The morphogenic response was direct from all types of leaflet explants and varied only with respect to different plant growth regulators of the medium: 2,4-D combined or not with BA formed somatic embryo-like structures; NAA alone produced tubers and roots; BA alone resulted mainly in leaves; NAA combined with BA produced mainly roots. The intensity of the response varied accordingly to the explant type and orientation. Explants with petiolule or midrib from the basal part of the leaflet showed the highest morphogenic response compared to explants without petiolule or midrib or to explants from the apical part of the leaflet, in all the plant growth regulator combinations used. Explants with midrib from the basal part of the leaflet placed vertically into the media showed higher morphogenic response compared to those placed horizontally on the medium surface. With the objective to regenerate plantlets, explants were subcultured on MS with NAA and BA at various concentrations based on the explant response in the establishment medium, taking into consideration the initial explant type. The initial explant type did not affect the response in the subculture. Most plantlets (a tuber with roots and one leaf with one pair of leaflets) were produced by explants with embryo-like structures induced in a medium with only 2,4-D. Explants with tubers induced in a medium with NAA gave plantlets at 65–85% when subcultured in a medium with 4.44 μM BA alone or in combination with 2.69 μM NAA. Explants with leaves induced in a medium with BA and explants with roots induced in a medium with NAA and BA gave plantlets at low percentages (20–40%). The best response was produced by explants with embryo like structures induced in a medium with only 2,4-D which gave plantlets at 100% when subcultured in the medium with 2.69 μM NAA and 2.22 μM BA. Plantlets raised in different treatments were transplanted ex vitro after 22 weeks and exhibited 80–100% survival.     

Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot (Prunus mume) using immature cotyledons

This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 μM α-naphthaleneacetic acid (NAA) and 5 μM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted β-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated “Sirakaga” immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l⁻¹ kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses.     

Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers

Molecular characterization using RAPD analysis was carried out in eight cut flowers and two pot plant cultivars of chrysanthemum. Three of them (‘Refocus’, ‘Red Reagan’, and ‘Sheena Select’) were established in vitro and the occurrence of somaclonal variation was studied using the same molecular technique. Two induction media (MS + 0.1 mg l⁻¹ NAA + 0.1 mg l⁻¹ BA, and MS + 2.0 mg l⁻¹ IAA + 0.5 mg l⁻¹ Kinetin), and two proliferation media (MS + 0.1 mg l⁻¹ NAA + 0.2 mg l⁻¹ BA, and MS + 4.0 mg l⁻¹ IAA + 2.0 mg l⁻¹ Kinetin) were employed in order to evaluate the effect of the medium composition in the shoots’ stability. Likewise, the effect of the culture age was considered in assessing genetic stability. Monthly subcultures were carried out, identifying the origin and history of the shoots, throughout a nine-month proliferation period followed by acclimatization. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The use of RAPD for chrysanthemum cultivar characterization and somaclonal variation detection is discussed.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

The effects of explant and cytokinin type on regeneration of Prunus microcarpa

We investigated in vitro regeneration ability of Prunus microcarpa subsp. tortusa using various explants (root, cotyledon and hypocotyl pieces) and cytokinins [benzyladenine (BA), meta-Topolin (mT) and thidiazuron (TDZ)]. Sectioned cotyledon, root and hypocotyl pieces of in vitro grown seedlings were cultured on Nas and Read Medium (NRM) containing BA (7.5, 10, 12.5, 15 or 17.5 μM), mT (2.5, 5.0, 7.5, 10 or 12.5 μM) or TDZ (2.5, 5.0, 7.5, 10 or 12.5 μM). As a measurement of morphogenetic reaction, the ratios of regenerating explants and the numbers of primary adventitious shoots per regenerating explant were analyzed. Cotyledon explants exhibited higher regeneration ratios than hypocotyl explants, and the root explants were inappropriate for regeneration. Both BA and mT were effective on shoot regeneration but higher regenerating explant ratios were obtained when BA was used. In comparison with BA and mT, the effect of TDZ on enhancing explant regeneration ability was insignificant. Mean number of adventitious shoot per regenerating explant was between 1 and 4, and regenerating explant ratios were between 0% and 77%. The practical appliacations of the results are discussed.